The invention concerns a high-performance system for parallel and selective dispensing of extremely small volume micro-dropletsxe2x80x94of the order of a sub-multiple of nano-liter, even of a sub-multiple of pico-liter-, transportable cartridges and of a kit to put to practice such a dispensing, as well as the applications of this system, notably in chemistry, in biology, in biotechnology or pharmacyxe2x80x94in particular for the manufacturing of bio-chips, for pharmaceutical, immunological, or biochemical tests, for the screening of drug libraries or serum banks, for the preparation of drugs or their percutaneous administration by iontophoresis, or else in the fields of cosmetics, for the making of perfume atomisers or aerosol, in inkjet printing or in automobile electronics, specifically for gas or oil fuel injectors.
Recent research progress in genomics and proteomics have made available a considerable number of biological and therapeutical molecules for testing. Nevertheless, the low quantities of available biochemical products as well as their high cost have led to look for a sensible increase of experimentation abilities by performance optimisation.
In order to do so, various techniques have been used for handling biochemical liquids, such as colony transfer systems or micro-pipetting systems with piezoelectric actuators and, more recently, the inkjet printing technique.
The goal of these technologies is the manufacturing of bio-chips by dispensing biological probes (of oligonucleotides, proteins, peptides, etc.), pre-synthesised in the majority of cases, on a surface of different types of support, such as glass, nylon(copyright) or cellulose(copyright).
Known for example from documents U.S. Pat. No. 5,053,100 or U.S. Pat. No. 6,083,762, are dispensers using a piezoelectric transducer on a micro-dispenser. This technique allows the application in situ of pre-synthesised or synthesised oligonucleotides. However, these systems are made by the unit and are unfit for high-density parallel dispensing.
In document U.S. Pat. No. 6,028,189, dispensing of reagents is done through the use of inkjet-type micro-pumps, activated by a piezoelectric actuator. Each micro-pump is built in a silicon bloc, with droplets feeding and ejecting channels. In order to obtain syntheses in situ, four micro-pumps deliver DNA bases by ejection in circular wells, formed on a glass substrate having its motion controlled along two axes.
These systems do not allow a high performance application according to the present invention, that is a few hundred to a few thousand of droplets by cm2: with four pumps running at a few hundred of Hz, a machine according to the previous document would apply 100 000 droplets in a few hundred seconds. The synthesis of 25 oligonucleotides probes would then require more than 2 hours.
The invention aims at considerably increasing the performance of the selective dispensing of micro-droplets, through a strong increase of the density of parallel applications, comparable to the densities obtained by photochemistry for the stopping of specific molecules though photolithographic masks. This method is restricted to those molecules that can be fixed by photochemistry and it basically does not allow to handle the reagent droplets individually. Moreover, synthesis in situ, with this method, is restricted to about 25 mononucleotides.
Another objective of the invention is to allows a selectivity which is not only spatial, by distributing the reagent on predetermined or programmed sites, but also targeted, by distributing a selected one of a plurality of reagents on the predetermined site.
The invention also aims at creating a multifunctional system easy to adapt under different forms, for instance to biological and biochemical analysis miniature kits for enabling a great number of applications. In particular, the invention allows not to be restricted to the synthesis of 25 nucleotides per probe but to synthesise long probes reaching, for instance, 70 nucleotides, while keeping a high performance.
These objectives are met by using a dispensing head into which is made, through high density microelectronic-type techniques a matrix of wells of specific shape and fed according to a specific connection for a high-output selective dispensing.
More precisely, the object of the invention is a high-performance micro-droplets dispensing system, comprising a substrate covered by a membrane and means for deforming the membrane perpendicularly to each cavity formed in the substrate, and where the cavities, etched in a material forming the substrate, appear in the shape of wells crossing the substrate with a lateral continuous inner wall of axial symmetry; each well opens on the upper surface and the lower surface of the substrate as respectively a feeding opening and a duct opened as an ejection nozzle, the feeding opening presenting an opening higher than the nozzle of the duct, and the duct presenting a shape ratio between 1 to 20.
According to the invention, the expression xe2x80x9cdistributingxe2x80x9d is to be understood as the ejection or the suction of micro-droplets, by high-output of some tens to some thousands of micro-droplets dispensed parallely in a split second, the expression xe2x80x9caxial symmetry wallxe2x80x9d as a revolution or cylindrical surface, for instance of a square cross-section, and the expression xe2x80x9cshape ratioxe2x80x9d as the ratio between the height and the opening of the exit duct.
To reach this type of performance, the substrate shows a well density which can reach 10 000 per cm2, with a flow that can exceed 1 million droplets per second.
Advantageously, the substrate material is chosen among semiconducting materials such as silicon, gallium arsenide, silicon carbide, germanium, oxyde and insulating composites (such as SOI, initials for silicon-oxyde-insulator), glasses, silicon nitrides, polysilicon, ceramics, thermoplastic materials,xe2x80x94such as methyl polymethacrylate, polycarbonates, polytetrafluoroethylene, polyvinyle chloride or polydimethylsiloxane, thick photosensitive resins (for instance, the resin  less than  less than SUB  greater than  greater than )-, as well as metals, such as tungsten or stainless steels.
Preferably, Micro-manufacturing techniques to make the wells or the micro-channels are selected according to the material of the substrate:
wet or dry chemical etching such as reactive ion etching (RIE in short) or deep etching (D-RIE in short) for silicon and glass;
etching by spark-machining or electroforming for metals;
casting and polymerisation for thermoplastic materials;
photolithography, laser cutting, ultrasounds, or by abrasive projection for the majority of substrates.
According to preferred embodiments, the material of the membrane is selected among glass, silicon, elastomers and thermoplastics; the membrane may be etched using the above-mentioned etching techniques, in order to generate a network of well feeding micro-channels; these micro-channels are coupled at both ends to as least one feeding reagent tank.
According to particular embodiments, the means for locally deforming the membrane are embodied by electromagnetic, magnetostrictive or piezoelectric actuators. Alternative means are also considered such as thermal means of the inkjet type or thermoplastic by bimetallic type effect, the electro-vaporising generated by an electric field located between each well, or else electrostatic actuators.
All of the deforming means can be managed by a single command unit programmable through a multiplexing network. Such a unit allows to trigger simultaneously or successively the suction or the dispensing of identical or different reagents through all the wells, through blocs of wells or certain wells.
According to particular embodiments, the dispensing head has four or a multiple of four lines and a number of columns of wells adjusted to the required density, in order to carry out the synthesis of the DNA probes from the four basic nucleotides monomers for the production of bio-chips. The wells in each line are fed by the same reservoir through a micro-duct formed in the membrane parallely to the line of wells and coupled laterally to the line or orthogonally to the plane of the substrate, the reservoirs being etched in the membrane or placed at a distance and connected to the micro-ducts by flexible links.
The invention also concerns the applications of the dispensing head defined hereinabove. In order to implement at least some of them, it is more profitable to prepare a set ready for use in the form of:
dispensing cartridge including at least one dispensing head pre-filled with reagents and with titration plates of a semiconducting or thermoplastic material, that can show micro-bowls formed by etching of micro-electronic type, by machining, by moulding, by thermoforming, or any techniques adjusted to such a production;
dispensing kit including at least one dispensing head, that can be equipped with at least an suction pump and at least one titration plate, pre-filled with reagent or not.
These cartridges or these kits are especially intended for the preparation of bio-chips, by in situ synthesis or deposit of pre-synthesised oligonucleotides, for the collective or individual screening of biological molecules or of cells, for the preparation of drugs or for pharmaceutical tests or for immunological, biochemical and biological screening.
The percutaneous administration of drugs by iontophoresis can be made from a dispensing head as previously defined consisting of several wells. A system for applying an adequate potential difference on a piezoelectrical cell, or any other electromagnetic actuating means, form an administration device of a calibrated quantity of at least one drug contained or formed in this well.
Besides the applications above mentioned, the invention can also be used for separation and sorting, for instance in chromatography by selective filtration. Identical or different bio-cells are first fixed, grafted, blocked or hanged, by any known means, on the walls of the wells of a dispensing head according to the invention, for each well or block of wells. The dispensing head can be integrated at the tip of a syringe.